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    Materials and Methods in Biotechnology

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    Rohan Patel
    The document describes the materials and methods used to characterize cotton hybrids and their parents. It details the experimental materials, equipment, procedures for DNA extraction, quantification, quality assessment, and molecular analysis including RAPD, ISSR, and SSR techniques.

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    © All Rights Reserved
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    Materials and Methods in Biotechnology

    Uploaded by

    Rohan Patel
    79 views11 pages
    The document describes the materials and methods used to characterize cotton hybrids and their parents. It details the experimental materials, equipment, procedures for DNA extraction, quantification, quality assessment, and molecular analysis including RAPD, ISSR, and SSR techniques.

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    importan for biotechnology students

    Original Title

    Materials and Methods in biotechnology

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    The document describes the materials and methods used to characterize cotton hybrids and their parents. It details the experimental materials, equipment, procedures for DNA extraction, quantification, quality assessment, and molecular analysis including RAPD, ISSR, and SSR techniques.

    Copyright:

    © All Rights Reserved
    Download as PDF, TXT or read online from Scribd
    Download as pdf or txt
    79 views11 pages

    Materials and Methods in Biotechnology

    Uploaded by

    Rohan Patel
    The document describes the materials and methods used to characterize cotton hybrids and their parents. It details the experimental materials, equipment, procedures for DNA extraction, quantification, quality assessment, and molecular analysis including RAPD, ISSR, and SSR techniques.

    Copyright:

    © All Rights Reserved
    Download as PDF, TXT or read online from Scribd
    Download as pdf or txt
    of 11

    MATERIALS AND

    METHODS

    MATERIALS AND METHODS


    The present investigation entitled Molecular Characterization of Desi Cotton
    Hybrids and Their Parents was carried out at the Main Cotton Research Station, Navsari
    Agricultural University (NAU), Athwa Farm, Surat. This chapter contains thedetails
    regarding the experimental materials and methodology adopted during the courseof
    investigation.
    3.1Experimental Material
    The experimental material of cotton genotypes were procured from the Main Cotton
    Research Station, Navsari Agricultural University, Athwa Farm, Surat. The details of
    genotypes included in the present investigation are summarized in Table 3.1.1
    Table No. 3. 1.1: List of hybrids and their parents used in the present study.
    HBRIDS

    FEMALE PARENT

    MALE PARENT

    G.cot.DH-7

    Sujay

    G.27

    G.cot.DH-9

    4011

    824

    3.1.2 Laboratory ware


    All glass ware used were obtained from Borosil and disposable plastic
    ware(centrifuge, tubes, PCR tubes, micro tips) from Eppendorf.
    3.1.3 Chemicals
    All the chemicals used were of analytical reagent grade/molecular grade and were
    obtained from Hi-Media/ E-Merck/Qualiges and Bangalore Genei.

    13 | P a g e

    3.1.4 Equipments:
    NAME OF INSTRUMENTS
    Weighing balance
    Laminar flow
    Camera
    pH meter
    Spectrophotometer
    Water bath
    Electrophoresis unit
    Micropipettes
    Centrifuge
    Hot Air Oven
    Gel documentation
    Thermo Cycler
    Dry bath

    COMPANYNAME
    SHIMADZU
    MAC, Delhi
    Sony
    GeNei
    EI
    GeNei
    GeNei
    Eppendrof
    Dynamica
    EIE
    UVTEC (Cambrige)
    BIORAD
    GeNei

    3.2 Extraction of plant genomic DNA


    Genomic DNA isolated from young fully expanded leaves ofhybrids and their
    respective parents using the CTAB method with minor modification. (Sanghai Maroof et al.,
    1984).
    Materials:

    CTAB extraction buffer

    Liquid nitrogen

    Chloroform: Isoamylalcohol (24:1)

    Isopropanol (prichilled at-20C)

    70% Ethanol

    T10E1

    3.2.1Procedure:

    100 mg of leaf tissue was taken from the young plant. The leaves were surface
    sterilized and washed with distilled water. The sample was ground to fine powder
    using liquid nitrogen with pre chilled pestle and mortar.

    The powder was transferred into a sterile 2.0 ml eppendorff tube. Then add 1 ml
    prewarmed CTAB extraction buffer [1M Tris (pH 8.0), 0.5 M EDTA (pH 8.0), 5 M
    NaCl, 0.01 M -mercaptoethanol, 3% CTAB, 1% PVP].

    14 | P a g e

    The tubes were vortexed and incubated at 65C for 1 hr with intermediate mixing.

    Equal volume of Chloroform: Isoamylalcohol (24:1) was added, mixed by mild


    vortexing for 5 to 10 sec and centrifuge at 13000 rpm for 15 min at room temperature.

    Supernatant (upper phase) was collected and add equal volume of Chloroform:
    Isoamylalcohol (24:1) in new tubes, mixed well and centrifuged at 15000 rpm for 15
    min at 4C.

    The top aqueous phase was transferred to another 1.5 ml tube and adds equal volume
    of ice cold isopropanol, mixed well and kept at -20C for 30-45 min.

    After this DNA was pelleted by centrifugation at 15000 rpm for 15 min at 4C.

    Discard supernatant add 500l of 70% ethanol. Again centrifuge at 14500 rpm for 3
    min at room temperature.

    Pellet was recovered, air dried and redissolved in 100 ml T10E1 buffer.

    Then add 2l of RNase (10 mg/ml) to the DNA tube and incubated at 37C in water
    bath for 30 min.

    Add equal volume of Chloroform: Isoamylalcohol (24:1) and centrifuged at 15000


    rpm for 15 min at 4C.

    The top aqueous phase was transferred to another 1.5 ml tube and equal volume of ice
    cold isopropanol, mixed well and kept at -20C for 30-45minutes.

    After this DNA was pelleted by centrifugation at 15000 rpm for 15 min at4C.

    The pellet was washed with 70% ethanol and centrifuge at 14500 rpm for 3 min at
    room temperature.

    Discard supernatant and pellet was recovered, air dried and redissolved in 100 ml
    T10E1 buffer.

    The DNA was stored at -20C for further use.

    3.3 Quantification of genomic DNA


    To estimate the quality and quantity (in terms of protein and RNA contamination) of
    isolated genomic DNA, spectrophotometry was performed. DNA (2l) was loaded in the well
    of Nanodrop instrument and the concentration of DNA and absorbance was taken at 260 to

    15 | P a g e

    280 nm were measured as well as the A260 /A280 ratio was calculated. The conversion factor or
    constant used was 50 for concentration measurement.
    3.4 Quality assessment of DNA by agarose gel electrophoresis
    Materials:

    Agarose powder

    1X TBE buffer

    Ethidium bromide

    Electrophoretic unit

    6X gel loading dye

    3.4.1 Procedure:
    The quality of DNA was assessment on 0.8% (W/V) agarose gel prepared in 100 ml
    1X TBE contains 3l Ethidium bromide (Etbr). The already extracted genomic DNA from
    stock (8l) was mixed with 2l of 6X gel loading dye. 7l sample was loaded in each well
    using micropipette. The gel was provided with potential difference of 100 V. The bands were
    visualized under UV light using transilluminator. The sample which resolved into single
    discrete high molecular weight band near the well with no shearing, were considered to be of
    good quality.

    3.5 Dilution of DNA


    For PCR reaction, the extract DNA was diluted to 10 ng/l for RAPD and ISSR
    analysis and 60 ng/l concentrations for SSR analysis using TE buffer.
    3.6 Molecular analysis of DNA
    3.6.1 RAPD analysis
    The genomic DNA is subjected to PCR. A total of Twenty five oligonucliotide
    primers GeNei) were used for RAPD analysis. RAPD with 25l reaction volume were carried
    out in 200l PCR tube. Each reaction mixture (25l) for PCR amplification consisted of 10X
    assay buffer (10mM Tris-HCl, pH 8.3 and 50mM KCl), 25mM MgCl2, 3U Taq DNA
    polymerase; 2.5 mM each dATP, dCTP, dGTP, dTTp (Banglore GeNei), 100ng/l decamer
    primer(Banglore GeNei), and 10ng genomic DNA template.

    16 | P a g e

    Table 3.2: Composition of master mix of RAPD


    Components

    Quantity

    Sterile distilled water

    17l

    10X assay buffer

    2.5l

    2.5mM MgCl2

    1l

    25mM dNTPs

    1l

    100ng/l primer

    1l

    3U/l Taq DNA polymerase 0.5l


    10ng/l Template DNA
    Total

    2l
    25 l

    Table 3.3: List of RAPD primers


    Sr.
    No.
    1
    2
    3
    4
    5
    6
    7
    8
    9
    10
    11
    12
    13

    Nameof primers

    Accession numbers

    RPI 1
    RPI 2
    RPI 3
    RPI 4
    RPI 5
    RPI 6
    RPI 7
    RPI 8
    RPI 9
    RPI 10
    RPI 11
    RPI 12
    RPI 13

    AM765819
    AM750044
    AM773310
    AM773769
    AM773770
    AM773771
    AM773312
    AM773773
    AM773315
    AM750045
    AM911709
    AM773316
    AM750046

    Sr.
    No.
    14
    15
    16
    17
    18
    19
    20
    21
    22
    23
    24
    25

    Nameof
    primers
    RPI 14
    RPI 15
    RPI 16
    RPI 17
    RPI 18
    RPI 19
    RPI 20
    RPI 21
    RPI 22
    RPI 23
    RPI 24
    RPI 25

    Accession
    numbers
    AM773774
    AM773775
    AM773776
    AM911710
    AM765830
    AM773777
    AM773317
    AM765820
    AM911711
    AM911712
    AM765821
    AM750054

    17 | P a g e

    3.6.1.1 PCR Programming for RAPD


    An initial denaturing step of 5 min at 94C, 30 cycles consisting each of a denaturing
    step of 45 sec at 94C, primer annealing step of 1 min at 35C and primer extension step of
    1min at 72C were given. Final step of 10 min at 72C was given for polishing the end of
    PCR products. PCR amplification was carried out in thermal cycler(BIORAD).
    3.6.1.2Electrophoresis of amplified DNA
    Amplified DNA fragments were separated on 1.5 % agarose gels and stained with
    ethidium bromide. Running buffer containing TBE [Tris-buffer, boric acid and EDTA(pH
    8.0)] was used for electrophoresis. Wells were loaded with 20 l of a sample and 5l of
    loading buffer (sucrose, bromophenol blue and Xylene cyanol). The gel was run at80 V
    (constant) to separate the amplified bands. The standard DNA marker (100 bp) was also run
    along with the samples. The separated bands were documented under UV transilluminator
    and photographed by Gel documentation system (AlphaInnotech.U.S.A.) and analyzed.

    3.6.2 ISSR analysis


    Nineteen oligonucleotide ISSR primers were used for hybrid analysis. ISSR with 25l
    reaction volume were carried out in 200l PCR tube. Each reaction mixture (25l)for PCR
    amplification consisted of 1X reaction buffer (10 mM Tris-HCl, pH 8.3 and 50mMKCl), 2.5
    mM MgCl2, 3U Taq DNA polymerase; 2.5 mM each dATP, dTTP, dCTP and dGTP (all
    reagents from Fermentas), 10 pico mole primer (Bangalore GeNei India),and 10 ng genomic
    DNA template.
    Table 3.4: Master mixture of ISSR
    Components
    Sterile distilled water
    10X assay buffer
    2.5mM MgCl2
    10mM dNTPs
    3U/l Taq polymerase
    10 p mol primer
    60 ng/l template
    Total

    Quantity(l)
    17
    2.5
    1
    1
    0.5
    1
    2
    25

    18 | P a g e

    Table 3.5: List of ISSR primers sequence


    Sr No.
    1
    2
    3
    4
    5
    6
    7
    8
    9
    10
    11
    12
    13
    14
    15
    16
    17
    18
    19

    Oligo name
    ISSR 1
    ISSR 2
    ISSR 3
    ISSR 4
    ISSR 5
    ISSR 6
    ISSR 7
    ISSR 8
    ISSR 9
    ISSR 10
    ISSR 11
    ISSR 12
    ISSR 13
    ISSR 14
    ISSR 15
    ISSR 16
    ISSR 17
    ISSR 18
    ISSR 19

    Sequence (5-3)
    AGCAGCAGCAGCAGCGA
    AGCAGCAGCAGCAGCGG
    AGCAGCAGCAGCAGCGT
    AGCAGCAGCAGCAGCGC
    CACACACACACACAAT
    CACACACACACACAAC
    CACACACACACACAGT
    CACACACACACACAGC
    CACACACACACACAGA
    CACACACACACACAAA
    GTGTGTGTGTGTGTTA
    GTGTGTGTGTGTGTTG
    GTGTGTGTGTGTGTCA
    GTGTGTGTGTGTGTCT
    GTGTGTGTGTGTGTAT
    GTGTGTGTGTGTGTAC
    CAGGAGAGAGAGAGAA
    GCTGAGAGAGAGAGAGA
    GCAGAGAGAGAGAGAGA

    Tm(C)
    48
    48
    48
    58
    46
    48
    48
    50
    48
    46
    46
    48
    48
    48
    46
    48
    52
    52
    52

    3.6.2.1 PCR programming for ISSR:


    An initial denaturing step of 5 min at 94C, 45 cycles consisting each of a denaturing
    step of 1 min at 94C, primer annealing step of 45 sec at 42C and primer extension step of 2
    min at 72C was given. Final step of 5 min at 72C was given for polishing the ends of PCR
    product. PCR amplification was carried out in Thermal cycler (biored). The amplified
    product was separated by electrophoresis and polymorphism was detected on 1.5% agarose
    gel by using ethidium bromide.
    Table 3.6: PCR amplification steps for ISSR
    Stage
    Initial denaturation
    Denaturation
    Annealing
    Extension
    Final extension
    Hold

    Tm (C)
    94
    94
    55
    72
    72
    4

    Duration
    5 min
    1 min
    45 sec
    45 sec
    10 min

    Cycles
    1
    40
    1

    19 | P a g e

    3.6.3 SSR Analysis


    A total 40 SSR primers, (MGHES, JESPR Series, Euro fins Genomics India Put Ltd)
    were used for the study. PCR reaction(25l) mixture contained DNA, 60 ng/l; 10X reaction
    buffer; 10mM dNTPs; 2.5mM MgCl2; 10 micromoles each of forward and reverse primers.
    The amplification was carried out in thermal cycler.
    Table 3.7: Master mixture of SSR
    Components

    Quantity (l)

    Sterile distilled water

    16

    10X assay buffer

    2.5

    2.5mM MgCl2

    25nm dNTPs

    10 p mol Forward primer

    10 p mol Reverse primer

    3U/l Taq polymerase

    0.5

    Template DNA

    Total

    25

    20 | P a g e

    Table 3.8: List of SSR primer sequencer


    Sr
    No.

    Primer
    name

    Reverse primer

    Forward primer

    Tm
    (C)

    1
    2
    3
    4
    5
    6
    7
    8
    9
    10
    11
    12
    13
    14
    15
    16
    17
    18
    19
    20
    21
    22
    23
    24
    25
    26
    27
    28
    29
    30
    31
    32
    33
    34
    35
    36
    37
    38
    39
    40

    JESPR-223
    JESPR-228
    JESPR-230
    JESPR-231
    JESPR-232
    JESPR-234
    JESPR-296
    JESPR-298
    JESPR-300
    JESPR-307
    JESPR-153
    JESPR-291
    TMB 409
    TMB 1268
    BNL 786
    BNL 3090
    CM 45
    CM 56
    NAU 2083
    MGHES 33
    MGHES 62
    MGHES 66
    JESPR -6
    JESPR-286
    JESPR-209
    TMB 283
    TMB 327
    TMB 1181
    TMB 1615
    TMB 1791
    CIR 61
    CIR 216
    BNL 1531
    BNL 1053
    BNL 1721
    BNL 3649
    BNL 1317
    BNL 1414
    BNL 686
    BNL 3408

    CGTTSCGGATTATTGGACATG
    GGCAAGCAAAGCAAAACTC
    GAAACCCTTGGCCATGAG
    CTATGAACTGCTGGCTATGG
    CGTTGTATTATTATTTCCAGTGCTCG
    CTAACTCGAATCCGTCAC
    TGACCTCAATTTAGAAACCC
    GGACCTTCGGAATAATTACC
    CGGAAAATGATGATGATGAGAAG
    GAAAGACACTAAGCTGAGGC
    GATTACCTTCATAGGCCA
    CATTCCCCACTTTGCTCT
    CAGAGGACGAAGGTAGC
    CAGGTACCATTGATGCCA
    CTTTCCACGTGTAATTTG
    GAAATCATTGGAAGAACA
    GATGCCAGTAAGTTCAG
    CAATCATAGACCGCCGCCACA
    AGAAGAGGTTGACGGTC
    TTTTTGGGCTTTCTTTTCT
    TGCATCTGATCTAATTGT
    TCCTCCTCCCACTTCATC
    ACTAAACCCTAAACACAA
    GGAGGACATGGGTTTGA
    ATTGAGAGGCATTTTGGT
    GGCTCCAAAATTGAAACG
    GCAATGTCTTCACCAG
    CATCTTCAATATTCACCAG
    TTGATTGATAACGCGACA
    GAGATCGTTTATCATTCC
    TTAGTCCTCTACATACCG
    ATCTGAACCATCATCCTC
    CTGCAACAAGAGCCTGT
    AGGGTCTGTCATGGTTG
    TGTCGGAATCTTAAGACC
    GCAAAAACGAGTTGACC
    AAAAATCAGCCAAATTGG
    AAAAACCCTTTCCAAGA
    ATTTTTCCCTTGGTGGTCCT
    ATCCAAACCATTGCACCACT

    TGGTCCAAAGCTCAAG
    CAGAACAACACCATCAACACTCTCAG
    GGGACTAAAGAAGTAATTATGCC
    GCTGGTGGGATTCTCTG
    CAGACCACGCTATTTTTGCC
    GCATAGTTATGAATGACTCTC
    GGGTGTTACATAGAGTGTATAAAATTG
    GATGCCCTCGTGTTAAAG
    CGCATCACAAACCAAACAC
    CTTGGCCATGTATTCCTTCA
    GAAAACATGAGCATCCTG
    CATGTTTCTTTGCCCATC
    TGGTGGGTTTCACTTTCA
    CTCGAAACCTAGTGC
    GATCTTAACTCTTGCTCT
    TTGCTCCGTATTTTCCAG
    GCCAACTTATATTCGGTT
    ACATTGCTAGATATTATA
    TGAGTGAAGAACCTGCA
    CCAATTACGCATGTTCAA
    TGTTCCTCACAGCAAGAG
    GACTGTGGCTGGAGGAG
    CATTATAAGGTCCCCAAT
    GCATGCATGTAAAATGTA
    AGATGACAAAAATTGTG
    GTGGACATTGGCATTCAT
    ATTGCCGACTTTGCCTGT
    ATTGCCGACTTTGCCTCT
    CAGCGTTGAACTATCCAC
    TCTACCGGCCTCGTAAG
    TCATAATAAAGGCGTGG
    TTCTGATTGGCACTTTC
    ATGGAGATTGGCTGAGAT
    CATGCATGCGTACGTGT
    GCGCAGATCCTCTTACCA
    CCTGGTTTTCAAGCCTGT
    CGTCAACAATTGTCCCAA
    GGTGTCCTTCCCAAAAATT
    ACATTGATAGAAATATAAACCAAACACG
    GTGTACGTTGAGAAGTCCA

    55
    55
    55
    55
    55
    55
    55
    55
    55
    55
    56
    56
    56
    56
    56
    56
    56
    56
    56
    55
    55
    55
    55
    55
    55
    55
    55
    55
    55
    55
    55
    55
    56
    56
    56
    56
    56
    56
    56
    56

    21 | P a g e

    3.6.3.1 PCR Programming for SSR


    An initial denaturation step of 5 min at 94C followed by 30 cycles consisting each of
    a denaturing step of 1 min at 94C, primer annealing step of 45 sec at 55C, and primer
    extension step of 1 min at 72C were given. The program ended with one final extension at
    72C for 10 min. The amplified products were resolved on 3% agarose gel with ethidium
    bromide.
    Table 3.9: PCR programming of SSR
    Stage

    Tm(C)

    Duration

    Cycles

    Initial denaturation

    94

    5 min

    Denaturation

    94

    1 min

    Annealing

    55

    45 sec

    Extension

    72

    45 sec

    Final extension

    72

    10 min

    Hold

    30

    22 | P a g e

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